Major trauma can cause volumetric muscle loss (VML) resulting in life-long disability. Although skeletal muscle is capable of remarkable regenerative potential, when injury is massive and destroys the underlying architecture, regeneration is aborted and is characterized instead by scar tissue formation. The standard of care in such injuries is wound closure, leaving little hope for functional recovery. The promise of regenerative medicine is the full regeneration of damaged tissues, either by promoting repair from endogenous stem cells or by the transplantation of cells to enhance regeneration. Perhaps the best example of this is skin grafting in the setting of massive tissue loss in burn victims. The fact that grafted skin contains endogenous stem cells assures that the graft will not only restore function acutely but also chronically as the stem cells function to replenish skin cells that are lost during the normal turnover of the tissue. Likewise, the long-term goal of regenerative medicine is to be able to restore damaged tissue and maintain that tissue for the full lifetime of the individual. Major advances have been made in the culture and transplantation of muscle stem cells (MuSCs, also known as ?satellite cells?) in recent decades, primarily in rodent models of muscle injury and degenerative disease. It has been known for over 40 years that transplanted myoblasts, the more differentiated progeny of MuSCs, can contribute to new muscle formation in the host. However, it has long been recognized that those cells have limited regenerative capacity and fail to form new stem cells. In our ongoing studies, supported by extensive Preliminary Data, we have been able to generate ?bioconstructs? that consist of decellularized muscle scaffolds into which we have engrafted MuSCs in a hydrogel. When this whole bioconstruct is transplanted into a VML lesion in a mouse hindlimb muscle, we are currently able to achieve limited structural and functional restoration. The major focus of the studies of this proposal is the development of this technology so as to optimize MuSC treatment of VML lesions and to design a scalable therapy that could be translated to humans. Toward this goal, we have outlined three Specific Aims, each based on extensive Preliminary Data: 1) To enhance MuSC therapy by generative bioconstructs that contain other cellular components of the MuSC niche so as to improve the engraftment and de novo muscle fiber formation by the MuSCs; 2) To optimize the use of physical activity in the form of voluntary running or forced treadmill running to enhance the efficacy of MuSC treatment of VML lesions; and 3) To assess our ability to scale up our model using at 10-fold increase in VML size and treatment with two separate approaches ? a direct scaling of our bioconstruct and the use of ?modular? bioconstructs. The overall goal of this proposal is to develop a scalable technology using MuSC bioconstruct transplantation for the treatment of VML. This will have direct and immediate relevance to Veterans who are suffering from skeletal muscle injuries, injuries that have limited their functional capacity and that, to date, have had no hope of further recovery. Our goal is to develop a novel therapeutic approach to muscle tissue repair based upon a deep understanding of the basic stem cell biology, a state-of-the-art application of bioengineering approaches to these clinical challenges, and a firm commitment to the clinical/translational mission to improve the health and quality of life of Veterans whose function and further rehabilitation is limited by the lack of effective therapeutic options.